本文選題：DNA　切入點：擴(kuò)增　出處：《西南大學(xué)》2014年碩士論文

【摘要】：一直以來,DNA都是分子生物學(xué)、生物化學(xué)、藥物分析等學(xué)科研究領(lǐng)域的明星分子。人們試圖通過努力多方位地認(rèn)識DNA分子,以便更好地利用DNA分子來改造客觀世界。因此,精確地分析DNA并挖掘其潛在的生物學(xué)功能很有研究價值。本文試圖建立一種簡單靈敏的短鏈DNA擴(kuò)增方法,并進(jìn)一步將DNA分子優(yōu)異的生物學(xué)性能用于材料組裝和靶向藥物運載中。具體研究內(nèi)容如下： (1)利用連接酶介導(dǎo)的聚合酶鏈?zhǔn)椒磻?yīng)(PCR)技術(shù)建立短鏈DNA的分析方法。PCR常用于臨床檢測病毒、細(xì)菌、細(xì)胞因子等的DNA或者RNA,但是通常只用于分析長鏈DNA序列,不能用于短鏈DNA的直接檢測。我們通過實驗設(shè)計了各自與靶物DNA一半互補的長度均為52個堿基的長鏈DNA作為檢測探針,在T4DNA連接酶的作用下,這兩段探針能以短鏈靶物DNA為模板連成104個堿基的長鏈,再將長鏈用PCR擴(kuò)增,就能實現(xiàn)對短鏈靶物DNA的間接放大檢測。借助DNA的特異性雜交作用以及PCR的擴(kuò)增作用,這段只有16個堿基的短鏈DNA的檢測限低至100fM,線性范圍跨越5個數(shù)量級,而且這種方法的抗干擾能力強,即使在復(fù)雜生物介質(zhì)細(xì)胞裂解液中也能靈敏地分析靶物DNA。 (2)利用雜交鏈?zhǔn)椒磻?yīng)(HCR)生成的長鏈DNA組裝金納米顆粒(AuNPs).納米材料組裝體因其微米尺寸而呈現(xiàn)出一些優(yōu)于單體的光電性質(zhì),成為了目前研究的熱點。雜交鏈?zhǔn)椒磻?yīng)是一種新的DNA自組裝技術(shù),它提供了一種合成長鏈DNA的簡單方法�；陔s交鏈?zhǔn)椒磻?yīng)生成的長鏈DNA,本文建立了一種簡單的納米材料的組裝方法,只需通過金納米顆粒與巰基DNA之間形成共價鍵,就能實現(xiàn)金納米顆粒沿長鏈DNA的組裝,其中長鏈DNA是通過雜交鏈?zhǔn)椒磻?yīng)生成的。通過控制雜交鏈?zhǔn)椒磻?yīng)的時間,可以得到長短不一的DNA產(chǎn)物,再與金納米顆粒作用后,生成各種長度的組裝體。研究發(fā)現(xiàn),這種鏈狀的金納米顆粒組裝體能清晰地在暗場顯微鏡下成像,而單個金納米顆粒的散射信號非常微弱。這表明,組裝后材料的光散射性質(zhì)大大提高。 (3)金納米顆粒介導(dǎo)的DNA負(fù)載阿霉素(Dox)用于癌癥的靶向治療。臨床癌癥治療中存在的主要問題有：藥物對正常組織的副作用大,藥物有效利用度低且耐受性差。因此建立有效的靶向給藥系統(tǒng),克服以上不足,對癌癥的治療很有意義。葉酸是一種普遍研究的靶向配體,而對于一些不表達(dá)或者少量表達(dá)葉酸受體的癌細(xì)胞,尋找一些其他的靶向配體用于提高藥物的療效有必要。我們通過在金納米顆粒表面修飾上含有朊蛋白核酸適配子的DNA用于負(fù)載阿霉素,建立一種靶向治療母細(xì)胞瘤的給藥系統(tǒng)。借助于核酸適配子與神經(jīng)瘤母細(xì)胞的特異性識別作用,導(dǎo)致DNA構(gòu)型發(fā)生改變,在癌癥部位釋放出阿霉素,特異性地殺死癌細(xì)胞。細(xì)胞毒性實驗和細(xì)胞熒光成像實驗很好地驗證了這種針對母細(xì)胞瘤的給藥系統(tǒng)的有效性。 綜上所述,本文首先建立了一種簡單快速的鏈?zhǔn)綌U(kuò)增短鏈DNA的方法,然后再利用DNA特殊的生物學(xué)性質(zhì)結(jié)合金納米顆粒優(yōu)良生物相容性及光電性質(zhì),將它們應(yīng)用于納米材料組裝及靶向藥物運載領(lǐng)域。本研究的優(yōu)勢在于：一是利用了PCR技術(shù)間接實現(xiàn)對短鏈DNA的靈敏分析：二是利用雜交鏈?zhǔn)桨l(fā)應(yīng)生成帶巰基的長鏈DNA,通過Au-S鍵的作用,將金納米顆粒組裝到DNA上；三是利用核酸適配子作為靶向配體,將阿霉素運送到神經(jīng)瘤母細(xì)胞中實現(xiàn)靶向治療癌癥。本研究為建立新的DNA分析方法及探索其進(jìn)一步應(yīng)用提供了參考價值。
[Abstract]:DNA has been a star in the fields of molecular biology , biochemistry , drug analysis , etc . It has been tried to make better use of DNA molecules to transform the objective world . Therefore , it is valuable to accurately analyze DNA and to explore its potential biological functions . This paper attempts to establish a simple and sensitive short - strand DNA amplification method and to further improve the biological properties of DNA molecules for material assembly and targeting drug delivery . The specific research contents are as follows :

( 1 ) Using ligase - mediated polymerase chain reaction ( PCR ) technique to establish a short - chain DNA analysis method . PCR is used to detect DNA or RNA of virus , bacteria , cytokines , etc . It is usually used to analyze long - chain DNA sequence and can not be used for the direct detection of short - strand DNA . By means of specific hybridization of DNA and PCR amplification , the detection limit of short - strand DNA of 16 bases is low to 100fM , and the linear range is over 5 orders of magnitude , and the method has strong anti - interference ability , and can be used to analyze target DNA sensitively even in complex biological medium cell lysate .

This paper presents a simple method for the synthesis of long - chain DNA by means of hybrid chain reaction .

( 3 ) Targeting therapy for cancer by gold nanoparticles mediated by adriamycin ( Dox ) . The main problems in the treatment of cancer are that the drug has a large side effect on normal tissues , low effective bioavailability and poor tolerance . It is necessary to establish an effective targeting ligand for cancer .

In conclusion , this paper first establishes a simple and rapid method to amplify short - strand DNA , then uses the special biological properties of DNA to combine the excellent biocompatibility and photoelectric properties of gold nanoparticles , and applies them to the field of nano - material assembly and targeting drug delivery . The research has the advantage that : firstly , the long - chain DNA with sulfydryl should be generated by means of PCR technique , and gold nanoparticles are assembled onto DNA by the action of Au - S bond ;
Third , using the nucleic acid aptamer as the targeting ligand , the adriamycin is transported to the neuroma mother cell to achieve the targeted treatment of cancer . The research provides a reference value for establishing a new DNA analysis method and exploring the further application thereof .

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R917

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 江臻q，

本文編號：1718618